A critical event in early atherogenesis is the retention of low density lipoprotein (LDL) particles in the subendothelium through their binding intimal proteoglycans. These retained lipoprotein particles are exposed to several modifying enzymes in the arterial wall, including lipases, oxidizing enzymes, and proteases. A multitude of biological responses to such modified LDL, including the recruitment and lipid loading of macrophages, leads to the initiation and progression of atherosclerosis. The extracellular matrix (ECM) appears to play an active role in this process by not only mediating the retention of LDL particles, but by also modulating the activity of various enzymes towards LDL. Thus, in regions where LDL is being accumulated, the co-localization of LDL, ECM and LDL modifying enzymes leads to a self-perpetuating cascade of events that culminates in atherosclerosis.
Within the arterial intima there are several proteolytic and lipolytic enzymes and oxidizing agents that are capable of modifying LDL. The activity of these factors is likely responsible for the prominent characteristics of LDL particles isolated from atherosclerotic lesions, namely oxidation, enrichment with sphingomyelin, and self-aggregation. Accumulating evidence points to LDL aggregation and fusion as elements of atherogenic lipid accumulation in the artery wall. Aggregated lipoproteins that appear to be derived from LDL are prominent in early atherosclerotic lesions. Aggregated LDL is taken up by macrophages in vitro at an enhanced rate compared to non-aggregated LDL, leading to macrophage cholesterol accumulation and foam cell formation. Since native LDL particles do not form aggregates, LDL modification appears to be a prerequisite for aggregation and fusion. It is notable that the aggregation/fusion of LDL particles proceeds faster in vitro when they are bound to proteoglycans, and in turn, proteoglycan binding of modified LDL is enhanced as a result of particle aggregation. Studies in vitro have demonstrated that treatment of LDL with sPLA2 and secretory sphingomyelinase (s-SMase), induces LDL aggregation and/or fusion and enhanced retention in the subendothelium (Oorni, K et al., J Lipid Res, 2000. 41(11):1703-14).
The phospholipases A2 (PLA2) family comprises a group of enzymes sharing the common feature of hydrolyzing the fatty acid esterified at the sn-2 position of glycerophospholipids. (Six, D. A. and B. A. Dennis, Biochem. Biophys. Act., 2000. 1488: 1-19). They are generally categorized into cytosolic or secretory forms. The secreted forms of the enzyme are of low molecular weight (14 kDA), highly enriched in disulfide bonds, and require 1-10 mM calcium for activity. The major secreted form present in synovial fluid, termed Group IIA (classically referred to as non-pancreatic secretory PLA2) has been proposed as a mediator of inflammatory responses. During acute or chronic inflammation, the concentration of Group IIA sPLA2 can increase by over 100-fold in inflammatory fluids and plasma. The expression of group IIA sPLA2 is widely distributed in human tissues. In the arterial wall, group IIA sPLA2 is expressed by vascular smooth muscle cells and is associated with heparin sulfate proteoglycans of the ECM
Recently, a distinct sPLA2 in macrophages and mast cells has been described. Although it was previously assumed that the sPLA2 in macrophages and mast cells is Group IIA, it is now recognized that Group V sPLA2 is expressed by these cells. Like the Group IIA enzyme, Group V sPLA2 mRNA is greatly induced by proinflammatory stimuli. Group V sPLA2 exhibits high affinity binding to proteoglycans that is mediated by a cluster of cationic residues near the C-terminal end of the enzyme. The studies of Balboa et al. demonstrate that an antisera raised against the synovial sPLA2 (Group IIA) cross-reacts with Group V sPLA2 in P388D1 macrophages. This observation underscores the importance of developing molecular probes and antibodies to distinguish the individual enzymes.
Immunohistochemistry studies have established that sPLA2 is present in normal arterial tissue and increased in atherosclerotic lesions. However, there are some inconsistencies between these reports. In some reports, sPLA2 is predominantly associated with smooth muscle cells, whereas in other reports, a significant amount is detected in macrophages. A possible explanation for the contradictory results may be the variable degree to which available antibodies recognize Group IIA sPLA2 and the related form, Group V sPLA2.
Group V sPLA2 shows a close structural relationship to Group IIA sPLA2. Nevertheless, these enzymes have distinct structural features that may exert important functional differences. Group V sPLA2 contains amino acid variations in the regions that have been shown to be important for the interfacial binding of other sPLA2s. Binding studies using phosphatidylcholine (PC)-coated hydrophobic beads showed that human Group V sPLA2 binds PC membranes more than 50-times more tightly than human Group IIA sPLA2. Indeed, it has been suggested that PC analogues may serve as selective inhibitors for Group V sPLA2, given the low binding affinity of other sPLA2s for PC substrates. In contrast to Group IIA sPLA2, which has been shown to be very ineffective in hydrolyzing PC compared to other phospholipids, kinetic studies show that Group V sPLA2 effectively hydrolyzes PC-containing substrates.
Thus, there is a need for probes and methods to distinguish the Group IIA and Group V sPLA2. There is also a need for methods and compositions to block LDL modifications that lead to LDL aggregation and/or fusion and increased macrophage LDL uptake.